International Journal of Systematic and Evolutionary Microbiology (2000), 50, 1081–1086
Printed in Great Britain
Phylogenetic analysis of the genus
Thermoactinomyc es based on 16S rDNA
sequences
Jung-Hoon Yoon and Yong-Ha Park
Author for correspondence : Yong-Ha Park. Tel : j82 42 860 4620. Fax : j82 42 860 4625.
e-mail : yhpark!kribb4680.kribb.re.kr
Korea Research Institute of
Bioscience and
Biotechnology (KRIBB),
PO Box 115, Yusong,
Taejon, Korea
The almost complete 16S rDNA sequences of the type strains of eight validly
described species and two invalid species of the genus Thermoactinomyc es
were determined and phylogenetically analysed. The validly described
Thermoactinomyc es species formed phylogenetic lineages related to the
family Bacillaceae, as shown previously. However, the available strains of
‘ Thermoactinomyc es glaucus ’ and ‘ Thermoactinomyc es monosporus ’ exhibited
their closest phylogenetic affinities not to the genus Thermoactinomyc es but
to the genus Saccharomonospora. Some Thermoactinomyc es species were
shown to be closely related from 16S rDNA sequence analysis. Particularly,
Thermoactinomyc es vulgaris KCTC 9076T and Thermoactinomyc es candidus
KCTC 9557T had the same 16S rDNA sequences and Thermoactinomyc es
thalpophilus KCTC 9789T and Thermoactinomyc es sacchari KCTC 9790T showed
16S rDNA similarity value of almost 100 %. From phylogenetic analysis based
on 16S rDNA sequences, it is suggested that the genus Thermoactinomyc es
should be taxonomically re-evaluated using other useful taxonomic markers.
Keywords : Thermoactinomyces species, 16S rDNA sequence, phylogeny
INTRODUCTION
The genus Thermoactinomyces was one of the earliest
known actinomycete taxa that was first proposed with
Thermoactinomyces vulgaris, the type species of the
genus (Tsiklinsky, 1899). There was no doubt in
recognizing Thermoactinomyces species as actinomycetes because of their morphological characteristics
of forming aerial and substrate mycelia. However,
some studies provided evidence that the genus Thermoactinomyces should no longer be classified within the
order Actinomycetales. Thermoactinomyces species
produce endospores as shown in bacilli (Cross et al.,
1968, 1971 ; Lacey & Vince, 1971) and have lower
GjC contents than those of actinomycetes (Lacey &
Cross, 1989). 16S rRNA oligonucleotide sequencing
revealed that the genus Thermoactinomyces is more
closely related to Bacillus species than to actinomycetes
(Stackebrandt & Woese, 1981). Park et al. (1993) also
showed that the type strain of Thermoactinomyces
vulgaris is phylogenetically related to the genus Bacillus
based on 5S rRNA sequences. Accordingly, it has been
.................................................................................................................................................
The GenBank accession numbers for the 16S rDNA sequences reported in
this paper are AF138732–AF138739, AF139879 and AF139880.
proposed that genus Thermoactinomyces should be
placed within the family Bacillaceae (Stackebrandt &
Woese, 1981 ; Park et al., 1993). Nevertheless, the
morphological characteristics of producing aerial and
substrate mycelia have led to classification of the genus
Thermoactinomyces becoming confused.
Thermoactinomyces species are aerobic, Gram-positive
and thermotolerant, with the exception of one
mesophilic species, Thermoactinomyces peptonophilis
(Nonomura & Ohara, 1971). The genus Thermoactinomyces contains meso-diaminopimelic acid but
no diagnostic sugars in the cell wall (Lacey & Cross,
1989), indicating that the wall chemotype is type III
(Lechevalier & Lechevalier, 1970). Differences in the
isoprenoid quinone profile of the genus Thermoactinomyces were seen between the studies of Collins et
al. (1982) and Tseng et al. (1990). Collins et al. (1982)
showed that this genus has unsaturated menaquinones
with seven or nine isoprene units (MK-7 or MK-9) as
the predominant menaquinones but Tseng et al. (1990)
found unsaturated menaquinones with seven, or eight
and nine isoprene units (MK-7 or MK-8 and MK-9) as
the predominant menaquinones. The genus Thermoactinomyces has a cellular fatty acid profile containing
major amounts of iso- and anteiso-branched fatty
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J.-H. Yoon and Y.-H. Park
acids (Kroppenstedt, 1985). The GjC contents of
DNA range from 52 to 54n8 mol % (Tm) (Lacey &
Cross, 1989). There are currently eight validly described Thermoactinomyces species, namely Thermoactinomyces candidus (Kurup et al., 1975), Thermoactinomyces dichotomicus (Krasil’nikov & Agre, 1964 ;
Cross & Goodfellow, 1973), Thermoactinomyces intermedius (Kurup et al., 1980), Thermoactinomyces
peptonophilus (Nonomura & Ohara, 1971), Thermoactinomyces putidus (Lacey & Cross, 1989), Thermoactinomyces sacchari (Lacey, 1971), Thermoactinomyces thalpophilus (Waksman & Corke, 1953 ;
Unsworth & Cross, 1980) and Thermoactinomyces
vulgaris (Tsiklinesky, 1899). Before including the eight
valid species, the genus Thermoactinomyces had very
confusing taxonomic history (Lacey & Cross, 1989). In
addition to the eight valid Thermoactinomyces species,
some species, such as ‘ Thermoactinomyces glaucus ’
(Henssen, 1957), ‘ Thermoactinomyces monosporus ’
(Waksman & Corke, 1953), ‘ Thermoactinomyces
thermophilus ’ (Waksman, 1961), ‘ Thermoactinomyces
antibioticus ’ (Craveri et al., 1964) and ‘ Thermoactinomyces albus ’ (Lacey & Cross, 1989), were known
but have not been validly described. ‘ Thermoactinomyces antibioticus ’ and ‘ Thermoactinomyces
albus ’ were described to be synonyms of Thermoactinomyces thalpophilus and Thermoactinomyces
vulgaris, respectively (Lacey & Cross, 1989). Strain(s)
of ‘ Thermoactinomyces thermophilus ’ are no longer
available from the culture collections. The type strains
of ‘ Thermoactinomyces glaucus ’ and ‘ Thermoactinomyces monosporus ’ are no longer available. However, one strain of ‘ Thermoactinomyces glaucus ’ and
one strain of ‘ Thermoactinomyces monosporus ’, which
are found in catalogues of some culture collections,
were those described by Fergus (1964) and Nonomura
& Ohara (1969), respectively.
Thermoactinomyces species have also been noticed due
to their pathogenicity. They have been implicated as
causal agents in various forms of hypersensitivity
pneumonitis (extrinsic allergic alveolitis), especially
farmer’s lung disease and bagassosis (Pepys et al.,
1963 ; Lacey, 1971 ; Lacey & Cross, 1989). Such
disease are likely to appear in farmers that have been
exposed to mouldy hay and cereal grains in which
Thermoactinomyces species are known to be most
abundant. However, Thermoactinomyces species are
known to be found in a variety of natural sources, such
as soil, rivers, dairy products and marine sediments,
and even in humidifiers of air-conditioning systems
(Lacey & Cross, 1989).
Despite confusing taxonomic history and their importance as pathogens, it is surprising that useful
taxonomic methods being recently used, such as
phylogenetic analysis based on 16S rDNA sequences,
have not been applied to the genus Thermoactinomyces. The 16S rRNA sequences of three
Thermoactinomyces species, Thermoactinomyces vulgaris, Thermoactinomyces candidus and Thermoactinomyces dichotomicus, were previously determined,
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but these sequences, especially 16S rRNA sequence of
Thermoactinomyces dichotomicus, may contain many
unreadable nucleotides and are therefore of little value.
The aim of this study was to examine the 16S rDNA
nucleotide sequences as one useful taxonomic marker
for systematic study of the genus Thermoactinomyces.
These sequences were thought to be very useful for
inferring phylogenetic relationships between Thermoactinomyces species as well as between the genus
Thermoactinomyces and other related genera.
METHODS
Bacterial strains. Table 1 summarizes the strains used in this
study and the GenBank accession numbers for the 16S
rDNA sequences. All strains, except Thermoactinomyces
dichotomicus, Thermoactinomyces intermedius, Thermoactinomyces peptonophilus and ‘ Thermoactinomyces
glaucus’, were grown in shake flasks containing CYC broth
(Cross & Attwell, 1974). Thermoactinomyces intermedius
was grown in shake flasks containing Trypticase Soy Broth
and Thermoactinomyces dichotomicus and ‘ Thermoactinomyces glaucus ’ were grown in broth medium containing 0n1 % yeast extract, 0n1 % beef extract, 0n2 %
N-Z amine (type A) and 1 % sucrose (pH 7n3). Thermoactinomyces peptonophilus was grown in broth medium
containing 1n5 % starch, 1 % yeast extract and 0n05 % MgSO
in tap water (pH 7n6). All strains, except Thermoactinomyces%
peptonophilus, were grown at suitable temperatures between
45 and 55 mC. Thermoactinomyces peptonophilus was grown
at 35 mC. The broth cultures were checked for purity before
they were harvested by centrifugation.
Chromosomal DNA isolation. Chromosomal DNAs were
isolated by the method described previously (Yoon et al.,
1996).
16S rDNA sequencing and phylogenetic analysis. 16S rDNA
sequencing was performed as described previously (Yoon et
al., 1998). However, forward primer 373F (5h-AATGGGCGCAAGCCTGAT-3h ; positions 373–390 in Escherichia
coli 16S rRNA numbering) was replaced by reverse primer
704R (5h-TCTRCGNATTTCACCNCTAC-3h ; positions
704 to 685 in E. coli 16S rRNA numbering). In some cases
sequencing reactions were performed with dITP from the
DNA sequencing kit (Amersham) or the SequiTherm EXCEL II DNA sequencing kit (Epicentre Technologies) to
relieve compression artefacts.
The 16S rDNA sequences determined were aligned with
16S rRNA gene sequences of other strains by using 
 software (Thompson et al., 1994). Reference sequences
were obtained from the GenBank database with the following accession numbers : X67148 (Atopobium minutum
NCFB 2751T), X60640 (Bacillus stearothermophilus NCDO
1768T), X60646 (Bacillus subtilis NCDO 1769T), V00348 (E.
coli), AB007908 (Lactobacillus delbrueckii JCM 1002T),
M58837 (Lactococcus lactis ATCC 19435T), X60632 (Paenibacillus polymyxa NCDO 1774T), Z38003 (Saccharomonospora glauca DSM 43769T), Z38007 (Saccharomonospora
viridis NCIMB 9602T), AB002521 (Streptococcus pyogenes
ATCC 12344T) and AF002262 (Thermomonospora curvata
JCM 3096T). Gaps at the 5h and 3h ends of the alignment were
omitted from further analysis. Evolutionary-distance
matrices were calculated by using the algorithm of Jukes &
Cantor (1969) with the  program within the 
package (Felsenstein, 1993). A phylogenetic tree was con-
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Phylogeny of the genus Thermoactinomyces
Table 1. Thermoactinomyces species and strains used in this study and accession numbers
of 16S rDNA sequences
.....................................................................................................................................................................................................................................
KCTC, Korean Collection for Type Cultures, Taejon, Korea ; JCM, Japan Collection of
Microorganisms, Institute of Physical and Chemical Research, Saitama, Japan ; IFO, Institute for
Fermentation, Osaka, Japan ; ATCC, American Type Culture Collection, Manassas, VA, USA ;
CCRC, Culture Collection and Research Center, Taiwan.
Species
Thermoactinomyces candidus
Thermoactinomyces dichotomicus
Thermoactinomyces intermedius
Thermoactinomyces peptonophilus
Thermoactinomyces putidus
Thermoactinomyces sacchari
Thermoactinomyces thalpophilus
Thermoactinomyces vulgaris
‘ Thermoactinomyces glaucus ’
‘ Thermoactinomyces monosporus ’
Strain
Accession no.
KCTC 9557T
KCTC 3667T (lJCM 9688T)
KCTC 9646T (lIFO 14230T)
KCTC 9740T (lATCC 27302T)
KCTC 3666T (lJCM 8091T)
KCTC 9790T (lCCRC 13341T)
KCTC 9789T (lCCRC 12549T)
KCTC 9076T
KCTC 9645 (lIFO 12530)
KCTC 3673 (lIFO 14050)
structed by using the neighbour-joining method (Saitou &
Nei, 1987) as implemented within the  program of
the  package. The stability of relationships was
assessed by a bootstrap analysis of 1000 data sets by using
the programs , ,  and  of
the  package.
RESULTS
Almost complete 16S rDNA sequences of 10 species
attributed to the genus Thermoactinomyces were determined. The 16S rDNAs were amplified by PCR and
non-phosphorylated strands of PCR products, whose
5h-phosphorylated strands were selectively digested by
λ exonuclease, were used as templates for sequencing.
The 16S rDNA sequences determined in this study
correspond to the region between positions 28 and
1524 by comparison with E. coli 16S rRNA.
The levels of 16S rDNA similarity between the type
strains of validly described Thermoactinomyces species
were very broad, ranging from 90n8 to 100 %. High
levels of 16S rDNA similarity were found between
some Thermoactinomyces species (Fig. 1). In particular, Thermoactinomyces vulgaris KCTC 9076T and
Thermoactinomyces candidus KCTC 9557T shared
identical 16S rDNA sequences. The two species were
shown to have 22 bp sequence differences and 10 gaps,
except ambiguous nucleotides, between their 16S
rRNA sequences determined previously. Thermoactinomyces intermedius KCTC 9646T also exhibited
relatively high 16S rDNA similarity value of 99n4 %
with Thermoactinomyces vulgaris KCTC 9076T and
Thermoactinomyces candidus KCTC 9557T. Thermoactinomyces sacchari KCTC 9790T and Thermoactinomyces thalpophilus KCTC 9789T had the same 16S
rDNA sequences, except a single position corresponding to one ambiguous nucleotide (C or T) of
Thermoactinomyces sacchari KCTC 9790T. Thermoactinomyces peptonophilus KCTC 9740T exhibited the
AF138732
AF138733
AF138734
AF138735
AF138736
AF138737
AF138738
AF138739
AF139879
AF139880
lowest levels of 16S rDNA similarity (90n8–91n8 %)
with other validly described Thermoactinomyces
species. This phylogenetic distinctiveness of Thermoactinomyces peptonophilus KCTC 9740T may have
been guessed, considering that Thermoactinomyces
peptonophilus KCTC 9740T has some physiological
characteristics different from those of other Thermoactinomyces species (Lacey & Cross, 1989). The 16S
rDNA sequences of two invalid Thermoactinomyces
species, ‘ Thermoactinomyces glaucus ’ KCTC 9645 and
‘ Thermoactinomyces monosporus ’ KCTC 3673, were
also compared with those of other Thermoactinomyces
species. ‘ Thermoactinomyces glaucus ’ KCTC 9645 and
‘ Thermoactinomyces monosporus ’ KCTC 3673 had
only a 1 bp sequence difference in their 16S rDNA
sequences, but they exhibited very low 16S rDNA
similarity values (less than 83 %) with the type strains
of validly described Thermoactinomyces species. The
phylogenetic analysis showed that ‘ Thermoactinomyces glaucus ’ KCTC 9645 and ‘ Thermoactinomyces
monosporus ’ KCTC 3673 cannot be members of the
genus Thermoactinomyces (Fig. 1). ‘ Thermoactinomyces glaucus ’ KCTC 9645 and ‘ Thermoactinomyces
monosporus ’ KCTC 3673 exhibited the highest 16S
rDNA similarity values with the genus Saccharomonospora, especially with Saccharomonospora glauca.
The 16S rDNAs of ‘ Thermoactinomyces glaucus ’
KCTC 9645 and ‘ Thermoactinomyces monosporus ’
KCTC 3673 showed only 1 bp and 2 bp sequence
differences, respectively, with 16S rDNA of the type
strain of Saccharomonospora glauca. The phylogenetic
tree was constructed using 16S rDNA\16S rRNA
sequences of Thermoactinomyces species determined,
the representatives of the family Bacillaceae, some
related taxa and some actinomycete species (Fig. 1).
The phylogenetic tree showed that the type strains of
validly described Thermoactinomyces species form a
distinct radiation of the cluster encompassed by the
genus Thermoactinomyces (Fig. 1). The tree indicates
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J.-H. Yoon and Y.-H. Park
.................................................................................................................................................................................................................................................................................................................
Fig. 1. Phylogenetic tree showing the positions of Thermoactinomyces species and representatives of some other taxa
based on 16S rDNA sequences. The scale bar represents 1 nucleotide substitution per 100 nucleotides. Bootstrap values
(expressed as percentages of 1000 replications) greater than 50 % are shown at the branch points. NCDO, National
collection of dairy organisms, Reading, UK.
that the genus Thermoactinomyces are much more
phylogenetically related to the family Bacillaceae than
to the actinomycetes, as shown in previous studies
(Stackebrandt & Woese, 1981 ; Park et al., 1993).
DISCUSSION
A phylogenetic study based on 16S rDNA sequences,
together with chemotaxonomic and genomic analyses,
is one of the most useful methods for inferring the
relationships between genera or between species belonging to a genus (Vandamme et al., 1996). However,
the genus Thermoactinomyces has scarcely been subjected to these methods and most species belonging to
this genus have been characterized by mainly relying
on morphological and physiological properties (Lacey
& Cross, 1989). Accordingly, 16S rDNA sequences
of the type strains of all valid species assigned to
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the genus Thermoactinomyces were determined and
phylogenetically analysed in the present study. Our
data confirmed previous findings that the genus
Thermoactinomyces is phylogenetically related not
to the actinomycetes but to the family Bacillaceae
(Stackebrandt & Woese, 1981 ; Park et al., 1993). This
study also showed the interspecific phylogenetic
relationships of the genus Thermoactinomyces based
on 16S rDNA sequences that were not revealed
previously. Some species were found to be closely
related by having high levels of 16S rDNA similarity
between them, and some species exhibited relatively
low levels of 16S rDNA similarity with other Thermoactinomyces species (Fig. 1).
Based on the results of 16S rDNA sequence analysis,
Thermoactinomyces vulgaris KCTC 9076T, Thermoactinomyces candidus KCTC 9557T and Thermoactinomyces intermedius KCTC 9646T are shown to be
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Phylogeny of the genus Thermoactinomyces
closely related and, particularly, 16S rDNA sequences
of Thermoactinomyces vulgaris KCTC 9076T and
Thermoactinomyces candidus KCTC 9557T were the
same (Fig. 1). Since Thermoactinomyces candidus
KCTC 9557T was distinguished from Thermoactinomyces vulgaris KCTC 9076T by differences of
some physiological properties, it was proposed as a
new species of the genus Thermoactinomyces (Kurup et
al., 1975). However, Thermoactinomyces candidus was
regarded as a synonym of Thermoactinomyces vulgaris
in Bergey’s Manual of Systematic Bacteriology (Lacey
& Cross, 1989) and, therefore, not listed in the manual
but the thought has not been accepted. A very close
phylogenetic relationship was also found between
Thermoactinomyces thalpophilus KCTC 9789T and
Thermoactinomyces sacchari KCTC 9790T, which
show a 16S rDNA similarity value of almost 100 %
(Fig. 1). DNA–DNA relatedness is now recognized
as being the most important criterion for defining
species in current bacteriology (Wayne et al., 1987 ;
Vandamme et al., 1996). The current phylogenetic
definition of a species states that strains with approximately 70 % or greater DNA–DNA relatedness are
members of the same species (Wayne et al., 1987).
From the results of 16S rDNA sequence analysis,
DNA–DNA relatedness test is likely to be necessary
for determining exact taxonomic relationships between
some Thermoactinomyces species. It is apparent that
Thermoactinomyces dichotomicus KCTC 3667T and
Thermoactinomyces peptonophilus KCTC 9740T need
not necessarily be subjected to a DNA–DNA
relatedness test, because they show levels of nucleotide
similarity that are low enough for them to be placed as
distinct species within the genus Thermoactinomyces
(Stackebrandt & Goebel, 1994). Two invalid species,
‘ Thermoactinomyces glaucus ’ KCTC 9645 and ‘ Thermoactinomyces monosporus ’ KCTC 3673, exhibited
their closest phylogenetic affinities not to the genus
Thermoactinomyces but to the genus Saccharomonospora, especially Saccharomonospora glauca. However, chemotaxonomic characterizations are also necessary to finally confirm the reclassification of the two
species to the genus Saccharomonospora, since the
genera Thermoactinomyces and Saccharomonospora
are different in some chemotaxonomic properties
such as predominant menaquinone profile and wall
chemotype.
The results of the phylogenetic analysis exhibit some
correlation with some physiological properties and
predominant menaquinone profiles shown in the study
of Tseng et al. (1990). The cluster containing Thermoactinomyces vulgaris KCTC 9076T, Thermoactinomyces candidus KCTC 9557T and Thermoactinomyces
intermedius KCTC 9646T and the cluster containing
Thermoactinomyces putidus KCTC 3666T, Thermoactinomyces sacchari KCTC 9790T and Thermoactinomyces thalpophilus KCTC 9789T show different
predominant menaquinone profiles. The type strains
of Thermoactinomyces vulgaris, Thermoactinomyces
candidus and Thermoactinomyces intermedius were
shown to contain MK-7 as the predominant menaquinones (Tseng et al., 1990). The type strains of
Thermoactinomyces sacchari and Thermoactinomyces
thalpophilus were shown to contain MK-8 and MK-9
as the predominant menaquinones (Tseng et al., 1990).
The predominant menaquinone profile for the type
strain of Thermoactinomyces putidus was not shown
but Thermoactinomyces putidus JCM 3213 has MK-8
and MK-9 as the predominant menaquinones (Tseng
et al., 1990). However, it should be considered that the
study of Collins et al. (1982) showed different menaquinone profiles from those shown in the study of
Tseng et al. (1990) for some Thermoactinomyces
species. Thermoactinomyces dichotomicus can be distinguished from other Thermoactinomyces species by
its morphological property of forming yellow to
orange colonies. The predominant menaquinone
profile of the type strain of Thermoactinomyces
dichotomicus was MK-7 in study of Collins et al. (1982)
but was not shown in study of Tseng et al. (1990). The
type strain of Thermoactinomyces peptonophilus forms
a line of descent distinct from other Thermoactinomyces (Fig. 1). It is mesophilic, unlike other
Thermoactinomyces species, and has some physiological properties distinguishable from other Thermoactinomyces species (Lacey & Cross, 1989 ; Nonomura
& Ohara, 1971). However, little is known about the
chemotaxonomic properties, including the menaquinone profile, of Thermoactinomyces peptonophilus
that may be necessary for investigating the taxonomic
relationships with other Thermoactinomyces species.
From the results of the phylogenetic analysis, together
with morphological and physiological properties and
predominant menaquinone profiles, it is supposed that
the genus Thermoactinomyces may be heterogeneous
group containing more than one genus. To solve this
question, a comparative taxonomic study using
additional phenotypic markers, especially chemotaxonomic markers, should be performed in the genus
Thermoactinomyces.
ACKNOWLEDGEMENTS
This work was supported by grants HS2321 from the
Ministry of Science and Technology (MOST) of the Republic of Korea. We are grateful to Dr Yong Kook Shin for
helpful discussion and to Dr Akio Seino for providing
Thermoactinomyces peptonophilus. We are also very grateful
to CCRC (Culture Collection and Research Center,
Taiwan), IFO (Institute for Fermentation, Osaka) and JCM
(Japan Collection of Microorganisms) for providing some
strains used in this study.
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